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ORIGINAL ARTICLE
Year : 2019  |  Volume : 32  |  Issue : 3  |  Page : 991-995

Differentiation of umbilical cord Wharton's jelly derived mesenchymal stem cells into cardiomyocytes using 5-azacytidine


1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Ministry of Health, Shebin El Kom Teaching Hospital, Menoufia, Egypt

Date of Submission02-Apr-2018
Date of Acceptance21-May-2018
Date of Web Publication17-Oct-2019

Correspondence Address:
Gehad H Shalby
Shebin El Kom, Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_136_18

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  Abstract 

Objectives
The objective of this article was to differentiate mesenchymal stem cells (MSCs) into cardiomyocytes.
Background
Cardiomyopathies resulted in permanent loss of cardiomyocytes as it had no ability of regeneration. This made MSCs a promising tool for cellular therapy because of their ability of self-renewal and multipotency. MSCs were considered ideal for cellular cardiomyoplasty as they can undergo full cardiogenic differentiation. Now it was believed that isolated cells from umbilical cord and expanded in vitro were a potential source of MSCs.
Patients and methods
An experimental study included 10 pregnant females due for delivery between October 2015 and April 2017. Umbilical cord samples and cord blood were collected from cesarean section patients, MSCs were cultured from umbilical cord tissue (Wharton's jelly). MSCs were subcultured in differentiating media containing azacytidine. Cardiomyocytes differentiation was detected by morphology of cardiomyocytes and immunophenotyping.
Results
MSCs were successfully isolated from 10 umbilical cord samples. MSCs showed positive expression of CD44 for umbilical cord mesenchymal stem cells (UCMSCs) mean ± SD (79.72 ± 5.85). It showed negative expression of CD34 for UCMSCs mean ± SD (1.08 ± 0.43). A significant statistical difference was found (P = 3.28 × 10−11) between MSCs and cardiomyocytes with respect to expression of troponin. A significant statistical correlation was seen (P = 0.001) between MSCs that showed positive expression of CD44 and cardiomyocytes.
Conclusion
By using azacytidine MSCs isolated from umbilical cord Wharton's jelly (UCWJ) can be differentiated into cardiomyocytes.

Keywords: azacytidine, cardiomyoplasty, mesenchymal stem cells, troponin, umbilical cord


How to cite this article:
Fathy WM, NourEldin RI, Shalby GH. Differentiation of umbilical cord Wharton's jelly derived mesenchymal stem cells into cardiomyocytes using 5-azacytidine. Menoufia Med J 2019;32:991-5

How to cite this URL:
Fathy WM, NourEldin RI, Shalby GH. Differentiation of umbilical cord Wharton's jelly derived mesenchymal stem cells into cardiomyocytes using 5-azacytidine. Menoufia Med J [serial online] 2019 [cited 2019 Nov 12];32:991-5. Available from: http://www.mmj.eg.net/text.asp?2019/32/3/991/268807




  Introduction Top


Cardiovascular diseases remained a leading cause of morbidity and mortality worldwide [1]. Congestive heart failure was the most common cardiac disease [2].

Cardiomyocytes loss was the final pathway to heart failure, which is caused by indifferent eating habits [3]. Treatment strategy of heart failure included nonpharmacological, pharmacological, and invasive strategies to limit and minimize its effects. The line of treatment was chosen according to the severity of illness [4],[5],[6].

Invasive therapies for heart failure included electrophysiological intervention, such as cardiac resynchronization therapy, pacemakers, and revascularization procedures that includes coronary artery bypass grafting, percutaneous coronary intervention, valve replacement or repair, and surgical operation [4],[5],[6],[7],[8].

Many varied therapies had been found to be of limited value, as the damage of myocardial wall was irreversible [9]. Heart transplantation was the standard therapy but high cost and shortage of donors was a serious problem [9].

Recently, cellular cardiomyoplasty with mesenchymal stem cells (MSCs), which were cells having the ability of differentiation into more specialized cells under certain stimuli, has been a new approach for repairing damaged myocardium [10].

MSCs has been isolated from nearly all tissues or organs, including umbilical cord blood and umbilical cord [11].

The aim of this study is to differentiate UCMSCs to cardiomyocytes using azacytidine.


  Patients and Methods Top


An experimental study was carried out at Clinical Pathology, Obstetrics and Gynecology Departments at Faculty of Medicine, Menoufia University during October 2015 to April 2017, after approval of the ethical committee of the hospital. It involved 10 pregnant females due for delivery, umbilical cord samples were used for Wharton's jelly and for cord blood serum collection. Samples were aseptically collected from cesarean section patients, after obtaining their consent. Under complete aseptic conditions umbilical cord was divided into small segments (about 6 cm each) and each segment was opened longitudinally. The umbilical cord (UC) vessels were dissected and removed. Wharton's jelly (WJ) was cut into small pieces of about 1.5–2.5 mm. Tissue culture plastic flasks 25 cm 2 (cell star) were prepared for culture by adding 5 ml of the fresh complete nutrient medium, which consisted of Dulbecco's modified Eagle's medium + l-glutamine (100 μg/ml) (Lonza, Basel, Switzerland)+cord blood serum 10%+penicillin–streptomycin (100 U/ml penicillin and 100 μg/ml streptomycin) (Lonza)+Fungizone (0.25 μg/ml) (Gibco, Co Dublin, Ireland). For proper adherence of the cells, the flasks were incubated in a horizontal position in an incubator with saturated humidity containing 5% CO2 at 37°C. At day 7, the tissue was removed by changing the medium. The flasks were washed twice with warm media, and the adherent cells (MSCs) were kept in culture and in complete nutrient medium (for about 1 week). These cells were kept until the outgrowth of fibroblast like cells. After removal of the tissue, the flasks were examined by the inverted microscope (×100 to × 200) for assessment of the cell morphology (viable cells were round, bright, and refractile). The media was examined every day, with assessment of the cell viability, and morphological changes. For 14 days, cells were examined microscopically to ensure 60–70% confluence; these cells were harvested by trypsinization. On day 14, for WJ, the harvested MSCs were identified by flow cytometric analysis of surface markers CD44 [phycoerythrin (PE)] and CD34 (PE). So, 100 μl of cell suspension was added to10 μl fluorochrome conjugated reagents (PE-conjugated anti-CD44). Another 100 μl of cell suspension was added to 10 μl fluorochrome conjugated reagents (PE-conjugated anti-CD34). Cells were incubated in darkness for 30 min after light vortexing. Finally, cells were analyzed by Flow Cytometer (Becton Dickinson FACS Calibur; Becton Dickinson: Franklin Lakes, New Jersy, US). The MSCs were gated out on CD44 and CD34 expression. Around 1 × 104 of MSCs were seeded in 35 mm culture dishes. Cells were treated with 10 μM 5-azacytidine (Sigma-Aldrich Co.: St. Louis, Missouri, US) on the second day after seeding. Media was changed every 3 days. The cells were allowed to differentiate over a period of 12 days. They were observed every day for any morphological changes by inverted, and phase-contrast microscope. Cells were regularly monitored using phase-contrast microscope and inverted microscope, images were captured for analysis. Samples were prepared for flow cytometric detection for intracytoplasmic detection of troponin by PE anti-human troponin T (R&D system, Minneapolis, MN, US) (fixation and permeability reagent purchased from Beckman coulter).


  Results Top


During first 3 days after the initial plating, cultured cells showed cytoplasmic projections by forming small clusters. On days 4 and 5, the onset of fibroblast could be observed. On day 9, cells tended to have a multipolar fibroblastoid appearance and a 60% confluence. By day 12 it increased to reach 80–90% confluence gradually.

Flow cytometric analysis of MSCs [Figure 1] showed positive expression for CD 44 (ranging between 72.20 and 88.10 with a mean ± SD of 79.72 ± 5.85), and showed negative expression for CD 34 (ranging between 0.50 and 1.70 with a mean ± SD of 1.08 ± 0.43) [Table 1].
Figure 1: Flow cytometric pictures representing positive expression of CD44 on MSCs, negative expression of CD34 on MSCs. MSC, mesenchymal stem cell.

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Table 1: Descriptive statistics of flow cytometric analysis results CD34, CD44, on mesenchymal stem cells

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Results of flow cytometric analysis of preinduction and postinduction UCMSCs: MSCs showed low positive expression of troponin with a mean ± SD of 1.12 ± 0.33. Whereas cardiomyocytes showed high positive expression of troponin with a mean ± SD of 74.12 ± 6.20 [Figure 2].
Figure 2: Flow cytometric pictures representing positive expression of troponin on induced cells (cardiomyocytes).

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A significant statistical correlation (P < 0.001) between MSCs and cardiomyocytes was found with respect to expression of troponin [Table 2] and [Figure 2], [Figure 3], [Figure 4], [Figure 5].
Table 2: Descriptive statistics of flow cytometric results of troponin

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Figure 3: MSCs before treatment with 5-azacytidine. MSC, mesenchymal stem cells.

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Figure 4: Cardiac bundles.

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Figure 5: Ball-stick like morphology.

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The cells were observed under ×10, ×20, and ×40 magnification. Cells were regularly monitored using phase-contrast microscope, and inverted microscope, images were captured for analysis [Figure 3].

MSCs induced with 5-azacytidine became thinner, and aligned parallel to each other. On day 6, they were seen groups of six to seven cells. They showed broadening of cytoplasm on day 7, whereas on day 9 some of them showed multinucleation extending their cytoplasmic processes with adjacent cells [Figure 4]. On day 12, they showed ball-stick like morphology [Figure 5].

Statistical analysis

Data were fed to the computer and analyzed using IBM SPSS software package, version 20.0 (IBM, Armonk, NY, US). The Kolmogorov–Smirnov test was used to verify the normality of distribution. Quantitative data were described using range (minimum and maximum), mean, SD, and median.

Student's t test was used for normally distributed quantitative variables, to compare between two studied groups.


  Discussion Top


Over the last years, stem cells had been found to be of great value in clinical research and regenerative medicine, due to their ability of self-renewal, and potential to differentiate into the various cell types of the organism [12].

Cellular cardiomyoplasty had the potential to improve healing of ischemic heart, restore injured myocardium, and cardiac function [13]. MSCs were progenitors of connective tissues, which had emerged as important tools for tissue engineering due to their differentiation potential along various cell types [14].

Our study aimed to study MSCs differentiation into cardiomyocytes using 5-azacytidine. UCMSCs were obtained from adequate number of patients undergoing cesarean section. The obtained cells were isolated and subcultured in differentiating media containing azacytidine. The produced cardiomyocytes were identified by their characteristic morphology, and by using immunophenotyping [10].

MSCs, known as multipotent mesenchymal stromal cells, are self-renewing cells that can be found in almost all postnatal organs and tissues [15].

In this study, we used cord blood serum (CBS) instead of fetal bovine serum (FBS) and the study reported CBS to be better for isolation of MSCs and that's insure 100% isolation of samples. This is in accordance with the study of Gul et al. [16], which showed that cells cultured in complete culture media-CBS (cord blood serum) had better growth in cultures as indicated by high number of population doublings.

In our study flow cytometric analysis revealed that MSCs showed positive expression for CD44 mean ± SD 79.72 ± 5.85 and negative expression of CD34 mean ± SD 1.08 ± 0.43. Also, in the study of Zhu et al. [17] human umbilical cord MSCs human umbilical cord mesenchymal stem cells (hUCMSCs) were isolated from the umbilical cords of normal or cesarean term deliveries under sterile conditions. Flow cytometry analysis revealed that CD13, CD29, CD44, CD90, and CD105 were highly expressed on the surface of passage-3 hUCMSCs, but negative for CD31, CD34, CD45, and human leukocyte antigen – DR isotype (HLA-DR).

This is in accordance with the study of Zhang et al. [18] who also investigated the differences between adipose-derived stem cells and MSCs in in-vitro culture and differentiation into cardiomyocytes. In their study, both adipose-derived stem cells and MSCs before treatment with 5-azacytidine were stained positively for CD29, CD44, and CD105 but negatively for CD34 and CD45, cardiac troponin T and von Willebrand factor.

Likewise, in our study it was found that after treatment with 5-azacytidine, the human umbilical cord-derived MSCs were morphologically transformed into cardiomyocyte-like cells, and expressed cardiac differentiation marker troponin (mean ± SD, 74.12 ± 6.20). During the differentiation, cells were monitored by a phase-contrast microscope, and their morphological changes were demonstrated. These observations confirm that human umbilical cord-derived MSCs can be transformed into cardiomyocytes and can be considered as a source of cells for cellular cardiomyoplasty [10].

MSCs did not express myocardial structural proteins before differentiation; troponin could be detected after differentiation. The positive expression rated of this protein was different depending on the differentiation conditions. The percentages of troponin positive cells were higher in the 5-azacytidine treated cells (42.8%) [19].

In this study, we aimed to differentiate UCWJ MSCs into cardiomyocytes using 10 μM 5-azacytidine. Media was changed every 3 days. The cells were allowed to differentiate over a period of 12 days.

Cultures of hMSC were treated with various concentrations of 5-azacytidine over 24 h. It was noticeable that 5-azacytidine did not exhibit any significant acute toxicity in the low micromolar range of concentrations, which were postulated to induce cardiogenesis [20].

In this study we use 10 μM 5-azacytidine which considered to be high concentration to prevent failure of differentiation of MSCs to cardiomyocytes. Cells were treated with 10 μM 5-azacytidine on the second day after seeding and media was changed as the 5-azacytidine showed toxic effect on the cells when used in high concentration.

The reason of failure of cardiac differentiation with 3 μM 5-azacytidine could be that the drug concentration used was too low in spite of longer duration. Some authors reported successful differentiation of hMSC with concentrations between 5 and 10 μM 5-azacytidine [21], suggesting that human cells may require higher concentrations than their murine counterparts, possibly because of higher activity of cytidine deaminase which degrades 5-azacytidine [22].

In this study MSCs induced with 5-azacytidine became thinner and aligned parallel to each other. On t day 6 they were seen in groups of six to seven cells. They showed broadening of cytoplasm on the day 7, whereas on day 9, some of them showed multinucleation extending their cytoplasmic processes with adjacent cells. On day 12 they showed ball-stick like morphology.

During exposure to 5-azacytidine, some cells died and got detached, whereas the surviving cells adhered and began proliferating and differentiating. One week later, ∼30–40% of the adherent cells in three experimental groups had enlarged, elongated, and formed stick-like morphologies. Within 2–3 weeks, the cells connected with adjoining cells, and formed myotube-like structures, branches, and string-bead-like nuclei. Some of the cells congregated into a cell cluster or cell strip. Under transmission electron microscopy, numerous myofilaments in the cytoplasm aligned in a parallel fashion were observed in all experimental groups but without forming typical striated sarcomeres, and conjunction of intercalated disc-like structure between adjoining cells was seen. There were no such histological changes in control cells [23].


  Conclusion Top


By using 5-azacytidin, MSCs can be differentiated into cardiomyocytes.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
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